N-acetylneuraminic acid links immune exhaustion and accelerated memory deficit in diet-induced obese Alzheimer’s disease mouse model

Systemic immunity supports lifelong brain function. Obesity posits a chronic burden on systemic immunity. Independently, obesity was shown as a risk factor for Alzheimer’s disease (AD). Here we show that high-fat obesogenic diet accelerated recognition-memory impairment in an AD mouse model (5xFAD). In obese 5xFAD mice, hippocampal cells displayed only minor diet-related transcriptional changes, whereas the splenic immune landscape exhibited aging-like CD4+ T-cell deregulation. Following plasma metabolite profiling, we identified free N-acetylneuraminic acid (NANA), the predominant sialic acid, as the metabolite linking recognition-memory impairment to increased splenic immune-suppressive cells in mice. Single-nucleus RNA-sequencing revealed mouse visceral adipose macrophages as a potential source of NANA. In vitro, NANA reduced CD4+ T-cell proliferation, tested in both mouse and human. In vivo, NANA administration to standard diet-fed mice recapitulated high-fat diet effects on CD4+ T cells and accelerated recognition-memory impairment in 5xFAD mice. We suggest that obesity accelerates disease manifestation in a mouse model of AD via systemic immune exhaustion.

This is an interesting manuscript that aims to understand how obesity induced by high fat diet (HFD) contributes to progression of AD and accelerates cognitive decline. To this end, 5XFAD mice were fed a HFD and compared to 5XFAD on control diet (CD) and WT mice on HFD and CD5XFAD on HFD. The manuscript shows that obese 5XFAD mice (with what appears to be 10-15% more weight than control mice) and did worse in the novel object recognition (NOR) test. Obese 5XFAD mice had similar Aβ and plaque loads but had worse neuronal loss than the other groups. To attempt to understand these findings, the manuscript shows that there is shrinkage of the naive CD4+ T-cells population and expansion of TEMs and Tregs, while TCRb+CD4 -T cells were unaffected in the spleen of obese 5XFAD mice. These mice also had the highest frequency of exhausted TEMs with decreased IFNg and TNF and increased PD1 and LAG3. To further understand the mechanism of such TEM exhaustion, polar metabolite analysis showed that NANA was increased and that the most likely source was the macrophages in visceral adipose tissues. Short term administration of NANA to middle aged WT mice led to increased CD4+PD1+ T cells and disrupts T cells metabolism. The manuscript concludes that NANA is the diet-associated metabolite associated with both the cognitive decline and the immune deregulation in obese 5xFAD mice, and that chronic antigenic stress and systemic inflammation, found in both obesity and AD, increased immune vulnerability to NANA in obese-AD mice.
Overall, the concepts advanced in the manuscript are relatively novel, and the manuscript is well written, with logical flow. Most of the experiments are well designed and the resulting data support most of the conclusions. However, the manuscript requires additional data to directly prove the most interesting point raised which is that NANA accelerates cognitive decline in obese 5XFAD mice via enhancing TEM exhaustion.

Following are my major concerns:
1-Only one test (NOR) is used to assess cognitive decline in 5XFAD mice in figures 1 and extended data Figure 2. This is hardly the norm. At least one additional test such as Morris water maze, radial arm maze or contextual fear conditioning should be performed to make the case. All of these are well established tests that work with 5XFAD mice.
2-it is not clear why frozen splenocytes were used for analysis of the peripheral immune system, why not using circulating blood immune cells. At lease the data from splenocytes should be validated by some analysis of circulating blood lymphocytes.
3-The administration of NANA to middle aged WT mice is interesting. However, the more direct and conclusive experiment is to administer NANA to 5XFAD mice fed control diet and show that the effects of HFD are reproduced by NANA administration including T cell exhaustion as well as cognitive decline and neuronal loss. Without this experiment the manuscript only shows that there is an association between obesity NANA, T cell exhaustion and accelerated neuronal loss and cognitive decline. This is the most relevant experiment that was not included in the manuscript and should be included. 4-It is not clear why peripheral immune exhaustion would cause neuronal loss and cognitive decline. In the absence of any experimental evidence, a detailed discussion is warranted and a cartoon explaining the pathway would be helpful (in addition to the experiments in point # 3 above) Minor point: 1-from how many mice (from each group) were the 269,578 nuclei that were included in the dataset derived?

Reviewer #3 (Remarks to the Author):
The authors present a manuscript entitled "Accelerated cognitive decline in obese mouse model of Alzheimer's disease is linked to sialic acid-driven immune deregulation" in which they demonstrate using a variety of confirmatory techniques that diet-induced obesity accelerated cognitive loss in 5xFAD mice. The authors also report that the comorbidity effect was functionally linked to systemic immune deregulation and that this was associated with diet-induced elevation of NANA. NANA or N-acetylneuraminic acid is a metabolite identified by the authors using an untargeted metabolomics platform to be inversely correlated with cognitive performance. I found the manuscript to be extremely well written and the inclusion of numerous confirmatory platforms refreshing.  In the manuscript entitled "Accelerated cognitive decline in obese mouse model of Alzheimer's disease is linked to sialic acid-driven immune deregulation" by Suzzi et al, the authors applied multiple state-of-the-art analytical technologies to assess the effects of high-fat diet on cognitive function, immune regulation (in different body compartments) and disease progression in 5xFAD mice. The study is overall very interesting and has potential impact on the field.
We thank the Reviewer for the overall positive feedback.
However, I have a number of major and minor concerns that should be addressed to improve the manuscript.

Major concerns:
Overall concept of the study: In summary, the authors have first demonstrated that HFD-induced obesity accelerated cognitive decline in 5xFAD, which was associated with changes in neuronal phenotype, increased neuronal loss, astrogliosis and changes in then cellular landscape of the hippocampus. No results regarding infiltrating immune cells (some were mentioned in the discussion) and specific changes in microglia population were mentioned here. Subsequently, the authors showed the findings on systemic immune deregulation after HFD in isolated splenocyte population. No information about how HFD affects the circulating immune cells in the peripheral blood, the population that are more likely to be in contact with the brain than do the splenocytes. Later, plasma metabolites were quantified, and the lipid-associated macrophages were identified as a source of NANA. This molecule was demonstrated to have influence on mouse splenocytes (in vivo and in vitro) and human blood cells. And no information about brain pathology in NANA-treated mice was shown. Due to this experimental design, several questions remain open and should be shown or (for some) at least discussed in the manuscript. These are for example: 1. How CNS immune cells (e.g. perivascular macrophages and microglia) are affected after HFD, and whether these can be related to changes in neuronal and astroglia population? The authors have briefly mentioned this by citing their own previous papers but the results from current study are missing.
We now included a more detailed description of the changes in the hippocampal cell landscape, including sub-clustering analysis of the major non-neuronal populations. We  Fig. 4a-f, 5a-g). Of note, for perivascular macrophages, we were able to capture this rare cell population in our sequencing data, and found a reduction in their frequencies in the 5xFAD model, but no significant additional change in their proportions with the diet (Fig.  2e, f). On the other hand, we found an increase in T cells specifically in the comorbidity model (Fig. 2e, f; Table 2).

differentially upregulated genes compared to all other immune cell clusters in Extended Data
2. Whether the phenotype and function of circulating immune cells in the peripheral blood (similar to splenocytes) are changed after HFD, and whether this is correlated with increased NANA in plasma?
We now added results of a CyTOF study in which we analyzed the effect of HFD on blood immune profile, and found that the major trend was the increase of CD4 + T cells irrespective of genotype (Extended Data Fig. 6a-c in the revised manuscript; relevant text at lines 137-140). This result led us to focus on lymphocytes, and on CD4 + T cells, in particular. In subsequent experiments we thus focused on the T-cell profile in the spleen as this organ is a major source of lymphocytes, and deregulation within the splenic CD4 + T-cell compartment was previously linked to aging 1,2 and neurodegeneration 3,4 (see text, lines 141-143).
3. It should be also possible to co-culture mouse microglia with NANA and assess the phenotypic or functional changes.
In our revised manuscript we added results showing that the microglia were marginally affected by the HFD (Fig. 2e, f, k; relevant text at lines 106-109 and 126-128). Moreover, we show that the levels of NANA in the hippocampus were not different across diet and genotype groups (Extended Data Fig. 10d; relevant text at lines 183-185). Based on these findings, we focused the study on the effect of NANA on T-cells and not on microglia. This is now clarified in the Results (lines 219-221).
3. Whether NANA treatment in WT or 5xFAD mice can induced similar brain pathology, observed in HFD-treated 5xFAD?
In the revised manuscript, we have results showing that repeated NANA administration led to accelerated loss of cognitive performance in the NOR test in 5xFAD mice (Fig. 6k, l;  Extended Data Fig. 16a-c; relevant text at lines 250-255). We therefore conclude that exposure to NANA exacerbated the disease phenotype in highly vulnerable mice (5xFAD mice). Please note that identifying the time window of high vulnerability, but when 5xFAD mice were not yet cognitively impaired, was a significant challenge that we overcame in this study. This is now highlighted in the Methods, lines 892-896.
Minor concerns: 4. Based on the results obtained from this study, one would be very curious to know whether NANA can cross the BBB? Or the immune modulation effects observed occur in the periphery and modulated immune cells are the one that drive changes in the CNS? This should be at least discussed.
NANA can enter the brain from the blood, but this amount is minimal, and the majority of it is cleaved by lyase activity 5 . As noted above (point 3), we measured NANA levels in the hippocampus, and found no difference across diet and genotype groups. Therefore, we hypothesized, based on our results, that the major effects of NANA on disease manifestation in 5xFAD mice is not due to its direct effect on the brain, but rather via its effect on the peripheral immune system. This is now clarified in the text, as suggested by the Reviewer (Discussion, lines 310-318). We also discuss a potential mechanism at lines 279-297. 5. The authors named "splenocytes" as "PBMCs" several time in the manuscript. To my understanding "splenocytes" are not "PBMCs" and possess quite different cellular composition and phenotypes.
We agree with the Reviewer and we now consistently refer to "splenocytes" where relevant throughout the text.
6. Line 417: …..To minimize inter-sample staining variability, sample handling time, and antibody consumption, cells were barcoded with Cell-ID 20-Plex (Fluidigm)…. This is not a strategy to minimize inter-sample staining variability.

Now rephrased (lines 503-505).
7. Since the authors used "targeted" approach for the quantification of metabolites, would it be possible that some metabolites, which are related to HFD and have strong effects will not be detected here?
It is indeed a possibility, since we performed targeted metabolite profiling based on a library of injected standards, which mainly contained polar metabolites, and did not investigate nonpolar metabolites. This point is now noted in the Discussion, lines 300-301. 8. Is there any specific reason, why sNUC-seq was used instead of scRNA-Seq?
In contrast to scRNA-Seq, sNuc-Seq can be used on tissues and cell types that cannot be easily dissociated. In order to get the full landscape of the cells of the hippocampus and visceral adipose tissue, we had to use nuclei, and not cells, because multiple cells are lost upon dissociation, while it remains possible to isolate their nuclei.

Reviewer #2 (Remarks to the Author):
This is an interesting manuscript that aims to understand how obesity induced by high fat diet (HFD) contributes to progression of AD and accelerates cognitive decline. To this end, 5XFAD mice were fed a HFD and compared to 5XFAD on control diet (CD) and WT mice on HFD and CD5XFAD on HFD. The manuscript shows that obese 5XFAD mice (with what appears to be 10-15% more weight than control mice) and did worse in the novel object recognition (NOR) test. Obese 5XFAD mice had similar Aβ and plaque loads but had worse neuronal loss than the other groups. To attempt to understand these findings, the manuscript shows that there is shrinkage of the naive CD4+ T-cells population and expansion of TEMs and Tregs, while TCRb+CD4 -T cells were unaffected in the spleen of obese 5XFAD mice. These mice also had the highest frequency of exhausted TEMs with decreased IFNg and TNF and increased PD1 and LAG3. To further understand the mechanism of such TEM exhaustion, polar metabolite analysis showed that NANA was increased and that the most likely source was the macrophages in visceral adipose tissues. Short term administration of NANA to middle aged WT mice led to increased CD4+PD1+ T cells and disrupts T cells metabolism. The manuscript concludes that NANA is the diet-associated metabolite associated with both the cognitive decline and the immune deregulation in obese 5xFAD mice, and that chronic antigenic stress and systemic inflammation, found in both obesity and AD, increased immune vulnerability to NANA in obese-AD mice.
Overall, the concepts advanced in the manuscript are relatively novel, and the manuscript is well written, with logical flow. Most of the experiments are well designed and the resulting data support most of the conclusions. However, the manuscript requires additional data to directly prove the most interesting point raised which is that NANA accelerates cognitive decline in obese 5XFAD mice via enhancing TEM exhaustion.
We thank the Reviewer for the positive comments and address the concerns raised below.
Following are my major concerns: 1. Only one test (NOR) is used to assess cognitive decline in 5XFAD mice in figures 1 and extended data Figure 2. This is hardly the norm. At least one additional test such as Morris water maze, radial arm maze or contextual fear conditioning should be performed to make the case. All of these are well established tests that work with 5XFAD mice.
In our initial studies we carried out also Radial Arm Water Maze. However, since the goal was to detect a time window in which the HFD accelerated cognitive loss in 5xFAD mice relative to the CD, we had to carry out repeated behavioral tests. We found that the NOR was the least stressful, and therefore continued with this assay throughout the entire work. In addition, we also assessed locomotor activity/anxiety by measuring the total distance moved and the time spent in the middle of the arena during the habituation trial of the NOR test. We now include these results in the revised manuscript (Extended Data Fig. 2d-g). We also report the total exploration time during the test trial (Extended Data Fig. 2b, c). The relevant text is at lines 64-79. The same parameters were tested in the additional data that we now include related to the effect of treatment with NANA on cognitive performance of 5xFAD mice (Extended Data Fig. 16a-c in the revised manuscript), as discussed below at point 3.

2.
It is not clear why frozen splenocytes were used for analysis of the peripheral immune system, why not using circulating blood immune cells. At lease the data from splenocytes should be validated by some analysis of circulating blood lymphocytes.
We now clarify the use of fresh splenocytes for general flow cytometry analysis (lines 143-144) and frozen splenocytes for mass cytometry (CyTOF) analysis (lines 152-154). We also now added CyTOF results of the blood immune profile that shows that the CD4 + T cells were impacted by the HFD, regardless of genotype (Extended Data Fig. 6a-c in the revised manuscript; relevant text at lines 137-140). Therefore, we focused in subsequent experiments on lymphocytes and CD4 + T cells, and thus analyzed the spleen as this organ is a richer source of lymphocytes, and splenic CD4 + T-cell rearrangements were previously shown to be linked to aging 1,2 and neurodegeneration 3,4 . With respect to validation of splenocyte data with blood lymphocyte data, we wish to emphasize that the ex vivo experiments, where we cultured mouse splenic T cells with or without NANA (Extended Data Fig. 13a-g in the revised  manuscript), were validated by the ex vivo experiments with human blood lymphocytes (Fig.  6a-c and Extended Data Fig. 14a-e in the revised manuscript). This is now clarified in the text (Results, lines 221-225; Discussion, lines 314-316).
3. The administration of NANA to middle aged WT mice is interesting. However, the more direct and conclusive experiment is to administer NANA to 5XFAD mice fed control diet and show that the effects of HFD are reproduced by NANA administration including T cell exhaustion as well as cognitive decline and neuronal loss. Without this experiment the manuscript only shows that there is an association between obesity NANA, T cell exhaustion and accelerated neuronal loss and cognitive decline. This is the most relevant experiment that was not included in the manuscript and should be included.
As we note in our reply to Reviewer #1, point 3, we now have results showing that 5xFAD mice treated with NANA, at 3-4 weeks after repeated NANA injections, displayed accelerated cognitive loss (Fig. 6k, l). In addition, in the same mice we show a CD4 + T-cell profile in the spleen and blood similar to the CD4 + T-cell profile observed in the spleen of middle-aged WT mice treated with NANA and analyzed 1 day after the last injection, which we reported in our original submission (Fig. 6h, j). In the revised manuscript, we describe these results at lines 250-267; the relevant Figures are Fig. 6h-l, Extended Data Fig. 15a-c, and Extended Data Fig. 16a-f. Finally, we wish to emphasize that both our ex vivo and in vivo experiments with NANA suggest that NANA has the potential to drive T-cell exhaustion, with CD4 + T cells being more susceptible (Fig. 6a-m; Extended Data Fig. 13a-g, Extended Data Fig. 14ae; Extended Data Fig. 15a-c). 4. It is not clear why peripheral immune exhaustion would cause neuronal loss and cognitive decline. In the absence of any experimental evidence, a detailed discussion is warranted and a cartoon explaining the pathway would be helpful (in addition to the experiments in point # 3 above).
There are numerous studies by our team and others, both in mouse models of aging and AD, and recently in humans, showing a correlation between progression of cognitive loss and reduction of IFN-γ production as well as elevation of Tregs in the periphery (for example, references 3,4,[6][7][8][9][10][11][12][13] ). This issue is now thoroughly discussed, and all the relevant citations are included in the revised manuscript, lines 279-297.
Minor point: 1. from how many mice (from each group) were the 269,578 nuclei that were included in the dataset derived?
The reported nuclei were derived from 28 animals, across all mouse genotype and diet groups (WT CD, WT HFD, 5xFAD CD, and 5xFAD HFD). Of note, in the revised manuscript we have 269,503 nuclei as few additional nuclei were removed due to their lower quality. The relevant text is in the Methods, lines 721-735.
Reviewer #3 (Remarks to the Author): The authors present a manuscript entitled "Accelerated cognitive decline in obese mouse model of Alzheimer's disease is linked to sialic acid-driven immune deregulation" in which they demonstrate using a variety of confirmatory techniques that diet-induced obesity accelerated cognitive loss in 5xFAD mice. The authors also report that the comorbidity effect was functionally linked to systemic immune deregulation and that this was associated with diet-induced elevation of NANA. NANA or N-acetylneuraminic acid is a metabolite identified by the authors using an untargeted metabolomics platform to be inversely correlated with cognitive performance. I found the manuscript to be extremely well written and the inclusion of numerous confirmatory platforms refreshing.
We thank the Reviewer for the appreciative comments.
I do have some concerns about the metabolomics platform used herein and the number of samples the authors used to come to their conclusions. I bring these comments up as a lot of their manuscript is reliant on the NANA story. I detail them below: 1. I would like to see a table listing all the "identified" metabolites in the extended data along with their relative concentrations. Further, I would also like to see the authors employ the Metabolites Standards Initiative scoring system for all the recorded metabolites. Table 3). Metabolites' abundance is reported as normalized peak area (Methods, lines 571-573).

We now provide as requested (Extended Data
2. I would suggest including the fragmentation pattern for NANA and also include that of the standard to show how they match up in terms of RT and M/Z. We now provide representative mass chromatograms and MS/MS spectra of NANA sample and standard (Extended Data Fig. 10a, b). Furthermore, in the revised manuscript we include targeted measurements of NANA levels in the hippocampus (Extended Data Fig.  10d), and provide representative LC-MS/MS chromatograms of NANA standard and 13 C 3 -NANA internal standard (Extended Data Fig. 10e).
2. I am very skeptical of the "n" used to identify NANA as being particularly important. The largest group is n=6 and for anyone who conducts metabolomics-based experiments, in particular untargeted metabolomics, having 4-6 biological replicates is very small indeed. I am not sure the correlation coefficients of NANA levels with NOR and Tregs are that strong either with the largest being 0.651.
As already reported in the original submission, we used a fluorometric assay to measure free NANA in a larger number of plasma samples, including samples from the same animals used for untargeted metabolomics (Fig. 4e in the revised manuscript). Using these measurements, we now show correlation of NANA levels with NOR discrimination index (Extended Data Fig. 10f) and splenic Tregs frequency (Extended Data Fig. 10g), in line with our findings from untargeted metabolomics ( Fig. 4c and d, respectively). The relevant text is at lines 1845-192.
3. Figure 3a, I would suggest including another box around "NANA". Maybe in blue, as it is hard to see from the figure which column you are referring to. Is there really that big of a difference across groups? Maybe it is my difficulty in discerning the column.
Based on the Reviewer's suggestion, we modified the relevant Figure (Fig. 4a in the revised  manuscript). In addition, we now show the amount of NANA per sample from untargeted metabolomics in Extended Data Fig. 10c. 4. I would also like to see if the results are recapitulated in another mouse model of AD. Not using all the experimental platforms but maybe a few.